A sensitive magnetic nanoparticle-based immunoassay of phosphorylated acetylcholinesterase using protein cage templated lead phosphate for signal amplification with graphite furnace atomic absorption spectrometry detection.

We developed a new magnetic nanoparticles sandwich-like immunoassay using protein cage nanoparticles (PCN) for signal amplification together with graphite furnace atomic absorption spectrometry (GFAAS) for quantification of organophosphorylated acetylcholinesterase adduct (OP-AChE), the biomarker of exposure to organophosphate pesticides (OPs) and nerve agents. OP-AChE adducts were firstly captured by titanium dioxide coated magnetic nanoparticles (TiO2-MNPs) from the sample matrixes through metal chelation with phospho-moieties, and then selectively recognized by anti-AChE antibody labeled on PCN which was packed with lead phosphate in its cavity (PCN-anti-AChE). The sandwich-like immunoreaction was performed among TiO2-MNPs, OP-AChE and PCN-anti-AChE to form TiO2-MNPs/OP-AChE/PCN-anti-AChE immunocomplex. The complex could be easily isolated from the sample solution with the help of magnet, and the released lead ions from PCN were detected by GFAAS for the quantification of OP-AChE. Greatly enhanced sensitivity was achieved because PCN increased the amount of metal ions in the cavity of each apoferritin. The proposed immunoassay yielded a linear response over a broad OP-AChE concentrations from 0.01 nM to 2 nM, with a detection limit of 2 pM, which has enough sensitivity for monitoring of low-dose exposure to OPs. This new method showed an acceptable stability and reproducibility and was validated with OP-AChE spiked human plasma.

我们开发了一种新的磁性纳米粒子夹心式免疫分析方法，该方法使用蛋白质笼状纳米粒子（PCN）进行信号放大，并结合石墨炉原子吸收光谱法（GFAAS）对有机磷酰化乙酰胆碱酯酶加合物（OP - AChE）进行定量。OP - AChE是接触有机磷农药（OPs）和神经毒剂的生物标志物。OP - AChE加合物首先通过与磷酸基团的金属螯合作用被二氧化钛包覆的磁性纳米粒子（TiO₂ - MNPs）从样品基质中捕获，然后被标记在PCN上的抗乙酰胆碱酯酶抗体选择性识别，PCN的空腔内填充有磷酸铅（PCN - 抗AChE）。在TiO₂ - MNPs、OP - AChE和PCN - 抗AChE之间进行夹心式免疫反应，形成TiO₂ - MNPs/OP - AChE/PCN - 抗AChE免疫复合物。借助磁铁可轻松从样品溶液中分离出该复合物，并且通过GFAAS检测从PCN释放的铅离子，从而对OP - AChE进行定量。由于PCN增加了每个脱铁铁蛋白空腔内的金属离子数量，因此实现了大幅提高的灵敏度。所提出的免疫分析方法在0.01 nM至2 nM的较宽OP - AChE浓度范围内产生线性响应，检测限为2 pM，这对于监测低剂量接触OPs具有足够的灵敏度。这种新方法显示出可接受的稳定性和重现性，并通过在人血浆中添加OP - AChE进行了验证。